Vaccination with plasmid DNA expressing antigen from genomic or cDNA gene forms induces equivalent humoral immune responses
Introduction
Genetic immunisation is a relatively new approach to vaccination, which mimics the manner by which live attenuated viruses deliver antigens, but without the associated risk of infection. Presently, genetic or DNA vaccination has been used to deliver viral [1], [2], bacterial [3], and parasite [4], [5] antigens to a variety of animal models from mice through to cattle [6] and sheep [4]. In vivo administration of plasmid DNA has also been used to generate monoclonal antibodies in the absence of purified protein, and as a tool to rapidly screen pathogen genomes for immunogenic and protective antigen genes via expression library immunisation [7], [8], [9]. However, while many examples of DNA vaccination have been reported, relatively little is still known about the immunobiological basis of DNA vaccination, and the optimal parameters for vaccine efficacy.
Investigations into the parameters affecting the efficacy of DNA vaccination have suggested the route of delivery used, the level of antigen expressed by transfected cells and whether antigen is maintained within transfected cells or secreted, can impact on both the quality and quantity of the immune response generated. Early studies of immune deviation revealed that more antigen is required to generate an antibody response than a DTH-type response [10]; this also appears to hold true for DNA vaccination [11] where the level of antigen expression correlates with the antibody response generated [11].
While the CMV promoter has now been well accepted as the optimal promoter for use in DNA vaccines, other construct modifications which can be employed to enhance in vivo expression of antigens include the introduction of a consensus eukaryotic ribosome recognition sequence such as that described by Kozak et al. [12]. The presence of introns in mRNA transcripts has also been shown to increase the expression of proteins in eukaryotes [11] and the optimisation of codon usage in antigen genes can significantly increase both cellular and humoral responses to DNA vaccines, presumably through enhanced mRNA translation [13].
While most DNA vaccines contain cDNA of antigen genes, genomic forms containing several internal introns may be available and the efficacy of cDNA or genomic forms of an antigen gene in DNA vaccines has not been directly compared. Similarly, while many subunit proteins produced as recombinant antigens are expressed in bacteria and hence are not glycosylated, the effect of glycosylated and nonglycosylated forms of antigen on the quality of the immune response elicited through vaccination is poorly understood. DNA vaccination with antigens containing glycosylation signals should generate glycosylated forms of antigen which can then be compared for immunogenicity with recombinant, nonglycosylated protein antigens.
Taenia ovis is a parasitic tapeworm, the metacestode stage of which causes ovine cysticercosis. The pathology of ovine cysticercosis results from cyst formation in muscular tissue, and the disease is of economic significance. Early studies into T. ovis found that vaccination with antigens from the oncosphere stage of the parasite could induce complete protection against infection [14]. The results of passive immunisation studies suggest that the mechanism of protection in sheep is via antibody [15]. Johnson et al. [16] identified a host protective antigen of 47–52 kDa which was designated 45W. The full length cDNA and genomic sequence of 45W was determined by Waterkeyn et al. [17]. The cDNA of full length 45W consists of a 765 bp open reading frame, while the genomic form of full length 45W comprises a 1731 bp open reading frame with 3 introns and 4 exons. Full length 45W is a 254 amino acid protein with a predicted molecular weight of 28 kDa, in contrast to the native form of the protein which migrates at 47—52 kDa in SDS-PAGE gels [17].
This study investigates the immunogenicity of the DNA vaccine pcDNA3-45W in Balb/c mice and outbred sheep and compares the immunogenicity of this prototypic 45W DNA vaccine [4], [18] with that of 45W DNA vaccines containing (1) cDNA or (2) genomic forms of the full length 45W gene with, an optimal ribosome recognition (i.e. Kozak) sequence around the AUG start. The in vitro expression of the constructs and the titre, kinetics and isotype of the antibody responses generated by these DNA vaccines was investigated. The fine peptide specificity of the antibody response generated by DNA vaccination was also compared with that generated by a conventional recombinant 45WB/X protein vaccine.
Section snippets
Vaccine plasmid construction
All vaccines were constructed using the DNA expression vector pcDNA3 (Invitrogen, CA, USA). This plasmid is 5.4 kilobases (kb) in size and genes inserted into the polylinker are under the control of the human cytomegalovirus (CMV) promoter. Three vaccine plasmids were generated in this study: pcDNA3-45W, pcDNA3-k45W and pcDNA3-kG45W. The construction of the plasmid DNA vaccine pcDNA3-45W has previously been described [4]. This vaccine contains a 985 base pair (bp) cDNA derived fragment encoding
In vitro expression of 45W antigen in transfected Cos7 cells
The ability of the vaccines to express full length antigen in vitro was investigated in Cos7 cells. DNA vaccines were transiently transfected into Cos7 cell monolayers using Lipofectamine Plus and the size of expressed antigen analysed by Western blots. Fig. 1 shows a Western blot of vaccine DNA (pcDNA3-45W, pcDNA3-k45W and pcDNA3-kG45W) and control DNA (pcDNA3) transfected Cos7 cells. Expression of 45W as a broad band of about 45–60 kDa in size was observed for all three vaccines tested. The
Discussion
DNA immunisation has attracted considerable interest for its technical simplicity, and for its capacity to generate complex immune responses. Recombinant protein forms of the 45W antigen generate high titre IgG1 and IgG2 antibodies in sheep [4], [18]. In contrast, sheep vaccinated with a DNA vaccine form of 45W develop only low titre antibody responses [4]. Sheep vaccinated with a 45W DNA vaccine and subsequently vaccinated with recombinant 45W protein [4], or adenovirus-encoded 45W [22],
Acknowledgements
Funding for this research was supplied by the Meat Research Council (MRC) of Australia and an Australian Postgraduate Award (APA) scholarship. Sheep vaccinations were performed with the assistance of Charles Goucci.
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